STRUCTURAL FEATURES AND LIGHT-DEPENDENT CHANGES IN THE CYTOPLASMIC INTERHELICAL E-F LOOP REGION OF RHODOPSIN - A SITE-DIRECTED SPIN-LABELING STUDY

Thirty consecutive single cysteine substitution mutants in the amino a cids Q225-I256 of bovine rhodopsin have been prepared and modified wit h a sulfhydryl specific nitroxide reagent. This sequence includes the E-F interhelical loop, a transducin interaction site. The accessibilit ies of the attached nitroxides to collisions with hydrophilic and hydr ophobic paramagnetic probes in solution were determined, and the elect ron paramagnetic resonance spectra analyzed in terms of side chain mob ility, both in the dark and after photoactivation. Accessibility data shows that the rhodopsin polypeptide chain crosses an aqueous/hydropho bic boundary in the range V227-K231 and again in the range V250-V254. In the hydrophobic segments, both the accessibility and mobility data are consistent with helical structures. In the regions of the sequence located within the aqueous phase, periodic variation in both accessib ility and mobility of the spin-labeled side chains indicates that the E-F interhelical loop is largely alpha-helical, being formed by regula r extensions of the E and F helices by about 1.5 and 3 turns, respecti vely. Judging from nitroxide mobilities, the putative extension of hel ix E in the aqueous phase is more dynamic than that of helix F. Change s in the electron paramagnetic resonance characteristics of the spin-l abeled rhodopsin upon photoactivation indicate that chromophore isomer ization results in patterns of structural changes that can be interpre ted in terms of movements of helices that extend into the aqueous loop regions.